Many of the mutations that underlie cystic fibrosis (CF) cause misfolding of the CFTR protein, making it dysfunctional or targeting it for degradation. Folding of CFTR and its insertion into the ER membrane is mediated by the translocon, a complex of proteins through which the nascent CFTR protein moves. Our hypothesis is that the translocon interacts with specific regions of CFTR transmembrane (TM) segments to direct protein integration into the ER membrane. A further hypothesis is that small sequence changes induced by point mutations alter CFTR-translocon interactions, leading to misfolding and degradation. To study the interactions of CFTR TM segments with the translocon, two specific aims will be pursued. In Aim I, photoactive cross-linking probes will be incorporated into CFTR-TM segments. When activated, these probes create covalent bonds with adjacent proteins. This allows one to define the portions of the CFTR protein that are in contact with the translocon during specific stages of protein processing. In Aim II, regions identified as important in Aim I will be studied in the same way in mutant CFTRs. Understanding CFTR biogenesis will allow a rational strategy for developing pharmacological approaches to CF defects.